Single protein-encapsulated pharmaceutics for enhancing therapeutic effects

ABSTRACT

The invention provides compositions comprising a single protein having one or more molecules of a pharmaceutical agent tightly bound therein. The compositions are useful to decrease the toxicity and/or to widen the therapeutic window of the pharmaceutical agent. The invention also provides methods for preparing such a composition.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.16/601,333, filed Oct. 14, 2019, which claims the benefit of U.S.Provisional Application Ser. No. 62/746,964, filed on Oct. 17, 2018. Theentire contents of these applications are hereby incorporated byreference herein.

BACKGROUND

Chemotherapy is a type of cancer treatment that uses one or moreanti-cancer drugs (chemotherapeutic agents) as part of a standardizedchemotherapy regimen. It comes to connote non-specific usage ofintracellular poisons to inhibit mitosis, cell division. Thechemotherapeutic agents could be either cytotoxic or genotoxic or both.Chemotherapeutic techniques have a range of side-effects that depend onthe type of medications used. The most common medications affect mainlythe fast-dividing cells of the body, such as blood cells and the cellslining the mouth, stomach, and intestines. Chemotherapy-relatedtoxicities can occur acutely after administration, within hours or days,or chronically, from weeks to years. In severe cases, chemotherapeuticagents can cause organ damages, such as cardiotoxicity (heart damage) byanthracycline drugs, hepatotoxicity (liver damage) by many cytotoxicdrugs, nephrotoxicity (kidney damage) by tumor lysis syndrome andototoxicity (damage to the inner ear) by platinum based rugs.

The small molecule anthracycline drug doxorubicin (DOX) is one of widelyused anticancer therapeutic agents. Among the most potent FDA-approvedchemotherapeutics, the anthracycline drug (Carvalho, C., et el., CurrMed Chem, 2009, 16, 3267-3285) displays a broad spectrum ofantineoplastic activities against both solid and hematologic tumors(Tacar, O., S et el., J Pharm Pharmacol, 2013, 65, 157-170; Tahover, E.,et el., Anticancer Drugs, 2015, 26, 241-258; and Shafei, A., et el.,Biomed Pharmacother, 2017, 95, 1209-1218). However, as a small andhydrophobic molecule, DOX indiscriminately infiltrates all tissues andorgans, causing systemic toxicities such as cardiomyopathy andmyelosuppression. When DOX's cumulative dose reaches a certain level,incidents of congestive heart failure increase sharply (Von Hoff, D. D.,et el., Ann Intern Med, 1979, 91, 710-717), which imposes a lifetimelimit of <450 mg/m² for DOX treatment. DOX-induced cardiomyopathy mayinvolve oxidative stress, contractile protein downregulation, andp53-induced apoptosis (Chatterjee, K., et el., Cardiology, 2010, 115,155-162). Its antitumor potency is derived from the fact that DOX caneasily penetrate cancer cell membranes and concentrate in the nucleus toeffectively bind DNA and subsequently inhibit topoisomerases, DNAreplication and transcription. At the same time, these properties alsoenable DOX to rapidly enter healthy cells in contact on its path,causing serious damage to healthy organs and tissues. In addition, DOXdisplays poor pharmacokinetics (PK) that can be described by eitherbiphasic (Greene, R. F., et el., Cancer Res, 1983, 43, 3417-3421; Speth,P. A., Linssen, et el., Cancer Chemother Pharmacol, 1987, 305-310; andSpeth, P. A., et el., Clin Pharmacol Ther, 1987, 41, 661-665) ortriphasic curves (Benjamin, R. S., et el., Cancer Res, 1977, 37,1416-1420; and Eksborg, S., et el., Eur J Clin Pharmacol, 1985, 28,205-212) with a short plasma circulation half-life of 5-12 minutes and aterminal phase half-life of about 30 hours (Speth, P. A., et el., ClinPharmacokine, 1988,t 15, 15-31). These undesirable characteristicsseverely impact the clinical outcome of DOX treatment due to its narrowtherapeutic window.

Various efforts and studies have been undertaken to reduce DOX'stoxicities. The key to success is to limit DOX's access to normal cellswhile increasing its traffic/delivery to tumors, which in principle maybe achieved by a rationally designed strategy ofassociating/binding/complexing/conjugating DOX with a macromolecular,self-assembled, or aggregated system with MW above the renal clearanceof about 50 kD. By associating DOX with a nano-sized moiety, such asystem significantly improves the PK of DOX, dramatically increases itscirculation lifetime, and enhances its access/delivery to tumors via theenhanced permeability and retention (EPR) effect due to tumors'irregular neovasculature and poor lymphatic drainage. Numerous studieswith liposomes, polymer conjugation, protein conjugation, proteinnanoparticles and metal/inorganic nanoparticles (NPs) have consistentlydemonstrated these underlying principles. However, current systems haveseen limited success, which is true even with FDA-approved Doxil(Petersen, G. H., et el., J Control Release, 2016, 232, 255-264). Thereare a number of reasons as to why current systems do not live up totheir expectations. Liposomes fuse nonspecifically with cell membranesto unload the cargo DOX, causing systemic toxicities. Chemicallyconjugating DOX to synthetic polymers and proteins modifies DOX to allowconjugation, but at the same time faces the challenge of its controlledrelease and changed properties due to chemical modifications. Whilemetal/inorganic NPs were once hailed as the silver bullets for efficientdrug delivery, they are far from natural systems. There is still not agood understanding of their interactions with tissues and organs.Furthermore, many of these non-natural systems may be recognized by ourbody's sophisticated immune system, leading to a broad range ofresponses that may vary among different patients. Lastly, there is alimited knowledge as to how the human body disposes an artificial system(synthetic polymer, metal/inorganic NPs, etc). While proteins andamide-based polymers may be enzymatically degraded to monomers that canbe used/processed by metabolism, it is unclear what the long termeffects are with non-biodegradable polymers and metal/inorganic NPs.Although human serum albumin nanoparticles (HSA-NPs) can have differentforms with varying sizes and chemical conjugation/crosslinking, they alldiffer significantly from free HSA (Kratz, F., J Control Release, 2008,132, 171-183; Sebak, S., et el., Int J Nanomedicine, 2010, 5, 525-532;Zensi, A., J Drug Target, 2010, 18, 842-848; Abbasi, S., J Drug Deliv2012, 686108; Elzoghby, A. O., et el., J Control Release, 2012, 157,168-182; Jin, G., et el., Oncol Rep, 2012, 36, 871-876; Lomis, N., etel., Nanomaterials, 2016, (Basel) 6; and Nateghian, N., et el., ChemBiol Drug Des, 2016, 87, 69-82). Consequently, their circulation PK,interaction with host organs/tissues/cells, and potential elicitation ofimmune responses can be considerably different from those of naturalHSA. All these issues directly contribute to the limited success withcurrent cancer drug formulation/delivery systems (Petersen, G. H., etel., J Control Release, 2016, 232, 255-264; van der Meel, et el., ExpertOpin Drug Deliv, 2017, 14, 1-5.; and Mukherjee, A., et el., 1996, AllAbout Albumin: Biochemistry, Genetics and Medical Applications. SanDiego, CA: Academic Press Limited). Thus, there is an urgent need forformulations that not only reduce the toxicity, but also enhanceefficacy in human clinical settings for DOX and other anticancer drugs.

HSA is the most abundant serum protein in the body, with a total ofabout 460 g distributing among the blood circulation, the lymphaticsystem and the extracellular/intracellular compartments (Peters, T.,1996, All About Albumin: Biochemistry, Genetics and MedicalApplications. San Diego, CA: Academic Press Limited). Its functionsinclude providing essential colloidal osmotic pressure, balancing plasmapH, and binding and transporting hydrophobic molecules such as fattyacids and bilirubin. HSA possesses some unique properties (Hoogenboezem,E. N., and Duvall, C. L., Adv Drug Deliv Rev, 2018, 130, 73-89): 1)being highly soluble and thermally stable, 2) capable of binding avariety of ligands with different binding affinity, 3) being endocytosedand transcytosed into and cross cells via receptors, 4) displaying anunusually long half-life of 19 days due to effective endosome recyclingby the neonatal Fc receptor (FcRn) and rescue from renal clearance viaMegalin/Cubilin-complexes (Chaudhury, C., J Exp Med, 2003, 197, 315-322;Anderson, C. L., et el., Trends Immunol, 2006, 27, 343-348; Chaudhury,C., et el., Biochemistry, 2006, 45, 4983-4990; and Kim, J., Bronson, etel., Am J Physiol Gastrointest Liver Physiol, 2006, 290, G352-360), 5)able to accumulate at tumor tissues due to EPR effects, and 6) beingpreferentially taken up and metabolized by cancer cells to serve asnutrients (Stehle, G., et el., Crit Rev Oncol Hematol, 1997, 26, 77-100;Commisso, C., et el., Nature, 2013, 497, 633-637; and Kamphorst, J. J.,et el., Cancer Res, 2015, 75, 544-553).

SUMMARY

Applicant has identified a method to tightly bind therapeutic agents(e.g. doxorubicin) within single proteins (e.g. albumin), whilesubstantially maintaining the properties of the single protein. Thismethod provides new compositions having lower toxicity and/or widertherapeutic windows.

In one embodiment, the invention provides a composition comprising asingle protein having one or more molecules of a pharmaceutical agenttightly bound therein.

The invention also provides a method to treat cancer in an animalcomprising administering to the animal a composition that comprises asingle protein having one or more molecules of an anti-cancer agenttightly bound therein.

The invention also provides a method to treat a bacterial or fungalinfection in an animal comprising administering to the animal acomposition that comprises a single protein having one or more moleculesof an antibacterial or antifungal agent tightly bound therein.

The invention also provides a method comprising: a) combining a firstsolution that comprises a pharmaceutical agent with a second solutionthat comprises a single protein, water, and a polar organic solvent toprovide a third solution; and b) stirring the third solution underconditions that allow one or more molecules of the pharmaceutical agentto become tightly bound within each single protein molecule. Theinvention also provides a composition prepared by a method of theinvention.

The invention also provides a pharmaceutical composition thatcomprises 1) a single protein with one or more molecules of apharmaceutical agent tightly bound therein and 2) a pharmaceuticallyacceptable carrier.

The invention also provides a composition as described herein for theprophylactic or therapeutic treatment of cancer.

The invention also provides the use of a composition as described hereinto prepare a medicament for treating cancer in an animal.

The invention also provides a composition as described herein for theprophylactic or therapeutic treatment of a bacterial infection.

The invention also provides the use of a composition as described hereinto prepare a medicament for treating a bacterial infection in an animal.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 : Topotecan's spectral changes before and after Encapsulation inPBS (pH=7.4), see Example 2.

FIG. 2 : Epirubicin's (EPI) spectral changes before and encapsulation inPBS (pH=7.4), see Example 3.

FIG. 3 : HSA-encapsulated epirubicin particle size distribution by DLS,see Example 3.

FIG. 4 : Idarubicin's spectral changes before and encapsulation in PBS(pH=7.4), see Example 3.

FIG. 5 : HSA-encapsulated idarubicin particle size distribution by DLS,see Example 3.

FIG. 6 : Vincristine's spectral change before and after encapsulation inPBS (pH=7.4), see Example 5.

FIG. 7 : Daunorubicin's UV spectral Changes before and afterencapsulation in PBS (pH=7.4), see Example 6.

FIG. 8 : HSA-encapsulated daunorubicin particle size distribution byDLS, see Example 6.

FIG. 9 : Mitoxantrone's spectral changes before and after encapsulationin PBS (pH=7.4), see Example 7.

FIG. 10 : HSA-encapsulated mitoxantrone particle size distribution byDLS, see Example 7.

FIG. 11 : Doxorubicin's UV spectral Changes before and afterencapsulation in PBS (pH=7.4), see Example 8.

FIG. 12 : HSA-encapsulated doxorubicin particle size distribution byDLS, see Example 8.

FIG. 13 : Amphotericin B's spectral changes before and afterencapsulation in PBS (pH=7.4), see Example 8.

FIG. 14 : HSA-encapsulated amphotericin B particle size distribution byDLS, see Example 8.

FIG. 15 : Clofazimine's spectral changes before and after encapsulationin PBS (pH=7.4), see Example 10.

FIG. 16 : Methotrexate's spectral changes before and after encapsulationin PBS, (pH=7.4), see Example 11.

FIG. 17 : HSA-encapsulated methotrexate particle size distribution byDLS, see Example 11.

FIG. 18 : Rifampicin's spectral changes before and after encapsulationin PBS (pH=7.4), see Example 12.

FIG. 19 , Vinblastine's spectral changes before and after encapsulationin PBS (pH=7.4), see Example 13.

FIG. 20 , Vinorelbine's spectral changes before and after encapsulationin PBS (pH=7.4), see Example 14.

FIG. 21 : UV spectral changes of doxorubicin in composition 15, seeExample 20.

FIG. 22 : Time profile of dialysis of free form of doxorubicin, HSA/DOXmixture and HSA-encapsulated DOX at different pH values, see Example 16.

FIG. 23 : Mean tumor volume changes vs Days of treatments by control,free Dox and Composition 7, see Example 17.

FIG. 24 : Mean tumor volume changes vs Days of treatments by control,free mitoxantrone and Composition 6, see Example 17.

FIG. 25 : % Body weight Changes (Normalized) vs Days of treatments bycontrol, free Dox and Composition 7, see Example 17.

FIG. 26 : % Body weight Changes (Normalized) vs Days of treatments bycontrol, mitoxantrone and Composition 6, see Example 17.

FIG. 27 : % Body weight Changes (Normalized) vs Days of treatments by3×(15 mg/kg) and 4×(20 mg/kg) of Dox equivalents in Composition 7, seeExample 18.

FIG. 28 : % Body weight Changes (Normalized) vs Days of treatments by5×(25 mg/kg) and 6×(30 mg/kg) of Dox equivalents in Composition 7, seeExample 19.

FIG. 29 : Diagram of Albumin-encapsulated Pharmaceuticals (drugmolecules).

FIG. 30 : HSA-fully encapsulated pharmaceuticals.

FIG. 31 : HSA-partially encapsulated pharmaceuticals.

FIG. 32 : HSA particle size distribution by DLS.

FIG. 33 : UV spectral changes of epirubicin in composition 16, seeExample 21.

FIG. 34 : UV spectral changes of doxorubicin in composition 18, seeExample 23.

DETAILED DESCRIPTION

In one embodiment, the invention provides a composition comprising asingle protein having one or more molecules of a pharmaceutical agent“tightly bound” therein. As used herein, the term “tightly bound” meansthat the molecule of the pharmaceutical agent is encapsulated within thesingle protein; the pharmaceutical agent is not covalently bounded tothe single protein either directly or through an intervening group. Inone embodiment, the molecule of the pharmaceutical agent is completelyencapsulated by the single protein (FIG. 30 ). In another embodiment,only part of the surface area of the molecule of the pharmaceuticalagent is encapsulated by the single protein (See FIG. 31 ). In anotherembodiment, one or more molecules of the pharmaceutical agent may becompletely encapsulated by the single protein and one or more moleculesof the pharmaceutical agent may only have part of its surface areaencapsulated by the single protein.

As used herein, the term “single protein” includes a single molecularspecies of a protein of both natural and synthetic origins, includingproteins isolated from both living organisms and bioengineered systems.Furthermore, the protein may contain other non-protein componentsthrough either covalent linkage or noncovalent interaction. In oneembodiment, the term does not include multimolecular species of aprotein, such as a dimer, trimer, oligomer, or multimer. In oneembodiment, the single protein is an albumin, a globulin, a fibrinogen,IgA, IgM IgG, or another human protein.

As used herein, the term “albumin” includes any albumin. In oneembodiment, the albumin is mammalian. In one embodiment, the albumin ishuman, cow, sheep, horse, or pig albumin. In one embodiment, albumin isnon-mammalian. In one embodiment, the albumin is prepared fromrecombinant techniques. In the compositions of the invention, thealbumin is not present in the form of particles, e.g. a nano-particle.Accordingly, the tightly-bound molecules of the pharmaceutical agent areencapsulated in pockets within the albumin structure, not within poresof an albumin nanoparticle.

As used herein, the term “globulin” includes any globulin. Globulins area heterogeneous group of large serum proteins, not including albumin,which are soluble in salt solutions. There are three principal subsetsof globulins, which are distinguished by their respective degrees ofelectrophoretic mobility: alpha globulin, beta globulin, and gammaglobulin. Non-limiting examples of various globulins include clottingproteins, complement, many acute phase proteins, immunoglobulins (Igs),and lipoproteins. In one embodiment, the globulin is mammalian. In oneembodiment, the globulin is human, cow, sheep, horse, or pig albumin. Inone embodiment, globulin is non-mammalian. In one embodiment, theglobulin is recombinant globulin. In one embodiment, the globulin is animmunoglobulin (Ig), such as an IgA, IgM, IgG, IgE or IgD antibody.

As used herein, the term “antibody” includes a single-chain variablefragment (scFv or “nanobody”), humanized, fully human or chimericantibodies, single-chain antibodies, diabodies, and antigen-bindingfragments of antibodies that do not contain the Fc region (e.g., Fabfragments). In certain embodiments, the antibody is a human antibody ora humanized antibody. A “humanized” antibody contains only the threeCDRs (complementarity determining regions) and sometimes a few carefullyselected “framework” residues (the non-CDR portions of the variableregions) from each donor antibody variable region recombinantly linkedonto the corresponding frameworks and constant regions of a humanantibody sequence. A “fully humanized antibody” is created in ahybridoma from mice genetically engineered to have only human-derivedantibody genes or by selection from a phage-display library ofhuman-derived antibody genes.

As used herein, the term “fibrinogen” includes any fibrinogen.Fibrinogen is a soluble glycoprotein present in blood plasma, from whichfibrin is produced by the action of the enzyme thrombin. In oneembodiment, the fibrinogen is mammalian. In one embodiment, thefibrinogen is human, cow, sheep, horse, or pig fibrinogen. In oneembodiment, fibrinogen is non-mammalian. In one embodiment, thefibrinogen is a recombinant fibrinogen.

As used herein, the term “polar organic solvent” includes solvents thatare miscible with water or partially dissolved in water. For example,the term includes water miscible solvents or water partially dissolvedsolvents. The term “polar organic solvent” includes:

-   -   (1) Water soluble alcohols: methanol, ethanol, isopropanol,        butanol, pentanol, t-butanols, etc    -   (2) Water soluble diols and triols, tetraols: ethylene glycol,        propylene glycol, glycerol, etc    -   (3) Water soluble aldehydes and ketones: acetone, butanone,        pentanones, hexanones, acetaldehyde, formyl aldehyde,        propionaldehyde, butyraldehyde, etc    -   (4) Water soluble nitriles: acetonitrile, propionitrile,        butanitrile, etc    -   (5) Water soluble polymers with low molecular weight:        polyethylene glycols, polypropylene glycols, etc    -   (6) Water soluble amides: DMF, dimethylacetamide,        dimethylpropanamide, etc,    -   (7) Water soluble ethers: diethyl ether, THF, dioxanes, etc    -   (8) All Other water soluble organic solvents: DMSO, etc

As used herein, the term “pharmaceutical agent” includes anypharmaceutically active agent that can be tightly bound within thesingle protein. In one embodiment, the pharmaceutical agent ishydrophobic. In one embodiment, the pharmaceutical agent is watersoluble at the desired pH values. In one embodiment, the pharmaceuticalagent is an anticancer agent, an antiinflammatory agent, a CNS agent, anantifungal agent, or an antibiotic agent. In one embodiment, thepharmaceutical agent is an anti-cancer compound. In one embodiment, thepharmaceutical agent is doxorubicin. In particular, the pharmaceuticalagent is water soluble at a pH from about −4 to about 20. In oneembodiment the pharmaceutical agent is water soluble at a pH from about0 to about 14. In one embodiment the pharmaceutical agent has limitedwater solubility at any pH values. In one embodiment the pharmaceuticalagent is doxorubicin, epirubicin, mitoxantrone, daunorubicin,vincristine, vinorelbine, vinblastine, topotecan, irinotecan,actinomycin D, idarubicin, methotrexate, pemetrexed, raltitrexed, SN-38,ixabepilone, eribulin, vindesine, camptothecin, paclitaxel, docetaxel,bendamustine, nelarabine, pirarubicin, clofarabine, valrubicin,chlorambucil, etc. In one embodiment, the pharmaceutical agent comprisesan amino group or amino groups. In one embodiment, the pharmaceuticalagent comprises a carboxyl acid group or carboxyl acid groups. In oneembodiment, the pharmaceutical agent comprises carboxyl acid group(s)and one amino group. In one embodiment the pharmaceutical agent is anantibiotic agent. In one embodiment the pharmaceutical agent isamphotericin B, clofazimine, rifampicin, chloramphenicol, tetracycline,or a fluoroquinolone antibiotic.

As used herein, the term “second pharmaceutical agent” includes anypharmaceutically active agent. In one embodiment, the secondpharmaceutical agent can be tightly bound within the single protein. Inone embodiment, the second pharmaceutical agent is hydrophobic. In oneembodiment, the second pharmaceutical agent is water soluble at thedesired pH values. In one embodiment, the second pharmaceutical agent isan anticancer agent, an antiinflammatory agent, a CNS agent, anantifungal agent, or an antibiotic agent. In one embodiment, thepharmaceutical agent is an anti-cancer compound. In one embodiment, thesecond pharmaceutical agent is doxorubicin. In one embodiment, thesecond pharmaceutical agent is docetaxel. In particular, the secondpharmaceutical agent is water soluble at a pH from about −4 to about 20.In one embodiment the second pharmaceutical agent is water soluble at apH from about 0 to about 14. In one embodiment the second pharmaceuticalagent has limited water solubility at any pH values. In one embodimentthe second pharmaceutical agent is doxorubicin, epirubicin,mitoxantrone, daunorubicin, vincristine, vinorelbine, vinblastine,topotecan, irinotecan, actinomycin D, idarubicin, methotrexate,pemetrexed, raltitrexed, SN-38, ixabepilone, eribulin, vindesine,camptothecin, paclitaxel, docetaxel, bendamustine, nelarabine,pirarubicin, clofarabine, valrubicin, chlorambucil, etc. In oneembodiment, the second pharmaceutical agent comprises an amino group oramino groups. In one embodiment, the second pharmaceutical agentcomprises a carboxyl acid group or carboxyl acid groups. In oneembodiment, the second pharmaceutical agent comprises carboxyl acidgroup(s) and one amino group. In one embodiment the secondpharmaceutical agent is an antibiotic agent. In one embodiment thesecond pharmaceutical agent is amphotericin B, or clofazimine, orrifampicin, chloramphenicol, or tetracycline, or fluoroquinoloneantibiotics.

HSA is well-known for its conformation changes when its environment isaltered. It has been reported that HSA displayed different confirmationsin acidic, neutral and basic conditions. HSA's conformation in acosolvent, such as ethanol/water, or methanol/ethanol/water, or 1,4-dioxane/water, or 2-butanone/ethanol/water, or acetone/water, orDMSO/water, or other organic solvents/water mixtures is dramaticallydifferent from the pure water (Borisover, M. D., et el., ThermochimicaActa, 1996, 284, 263-277). The literature shows that suspending HSA inthe water/organic coso vents is accompanied by two main processes, (1)the water desorption-sorption, (2) the non-sorption that is attributedto rupture of protein-protein contact, depending on the nature oforganic solvent and water content. Furthermore, the prepared HSAsolution in the water/organic cosolvents results in the increase in theaccessible surface areas, which has capacity to change the watersorption and calorific properties of the intended HSA suspension. HSA inthe water/organic cosolvents is no longer in its natural state; it ispartially denatured. Due to the fact that relative polarity of thecosolvent is lower than the pure water's, the resulting conformationchanges of HSA in the desired organics/water mixture would allow some ofthe hydrophobic pockets to be opened up, allowing pharmaceuticals agentsto be tightly bound or encapsulated into these hydrophobic pockets, seeFIG. 29 . For example, in Composition 7 below, multiple molecules of DOXare tightly bound inside each HSA molecule. In literature reports (Khan,S. N., et el., Eur J Pharm Sci, 2008, 35, 371-382; and Agudelo, D., etel., PLoS One, 2012, 7, e43814) wherein DOX and mitoxantrone werereversibly associated to human serum albumin (HSA), only 1 molecule ofDOX or mitoxantrone was reported to be associated with each HSA.Additionally, the UV spectra of the reversibly associated DOX andmitoxantrone showed no change from the corresponding unassociatedmaterials. In this invention, once doxorubicin or mitroxantrone wasencapsulated into a HSA molecules, a red-shifting of UV spectra fordoxorubicin or mitroxatrone was observed, See FIG. 11 for composition 7and FIG. 9 for composition 6. In some case, once being capsulated intoHSA molecule, an absorbance band of drug molecules is eliminated, seeFIG. 13 for composition 8 where an absorption at 324 nm of amphotericinB is completely eliminated one being encapsulated inside HSA molecule.In other case, the blue-shifting of UV spectra of certain drug moleculesafter being capsulated into a HSA molecule was observed and recorded,see FIG. 15 for composition 9, where clofazimine was encapsulated into aHSA molecule. The compositions of the invention that have pharmaceuticalagent molecules tightly bound within albumin have novel properties, suchas, for example, enhanced therapeutic effects.

In one embodiment, the single protein is dissolved in a co-solventscontaining at least one water soluble organic solvent that helps thepharmaceutical agent to be able for being encapsulated into the singleprotein. The encapsulation process is monitored by UV or otherinstruments. Once the desired percent of single protein-encapsulatedpharmaceuticals is achieved, the encapsulation process is terminated andthe final product is prepared. After the filtration (e.g. through 0.22um membrane or high speed centrifugation or other sterilizationprocedure), the concentrations of pharmaceuticals can be quantified byUV spectrometer, HPLC or other methods after organic solvent extractionthrough the proteins precipitation. After quantification, the singleprotein-encapsulated pharmaceuticals solutions can be frozen at −20° C.or lyophilized to powder products. In addition, in some embodiments, thesingle protein-encapsulated pharmaceuticals can be further purified viarunning through Sephadex G25 column, in which the large molecule, singleprotein-encapsulated pharmaceuticals come out the first, followed by theun-capsulated pharmaceuticals. In some embodiments, the singleprotein-encapsulated pharmaceuticals can be further purified viadialysis using the dialysis pouch with different molecular weightcutoffs, in which the un-capsulated pharmaceuticals with small molecularweights will be dialyzed out and the single protein-encapsulatedpharmaceuticals with macular weights >20 kd will be kept inside thedialysis bag. This invention provides novel method to prepare singleprotein-encapsulated pharmaceuticals without chemically modifyingstructures of the single protein or the intended pharmaceuticals.

HSA is a biopolymer, with a molecular weight at about 66,000 g/mole witha particle size at about 10 nm (FIG. 32 ) determined by dynamic lightscattering (DLS). In the compositions of the invention the particle sizeof the albumin having one or more molecules of a pharmaceutical agentencapsulated therein does not change, see FIG. 5 for composition 3, FIG.8 for composition 5, FIG. 10 for composition 6, FIG. 12 for composition7, FIG. 14 for composition 8, and FIG. 17 for composition 10. In oneembodiment the albumin having one or more molecules of a pharmaceuticalagent tightly bound therein is soluble in water. In another embodimentthe albumin having one or more molecules of a pharmaceutical agentencapsulated therein is soluble in water at a pH in the range of fromabout 6 to about 8.

If the pharmaceutical agent is an anticancer agent, the compositioncomprising a single protein having one or more molecules of thepharmaceutical agent encapsulated therein may increase the MTD of theagent, because the encapsulated molecules prefer to go to the cancercells and will have fewer interactions with the normal cells. If thepharmaceutical agent is an antibiotic, the composition comprising thesingle protein having one or more molecules of the pharmaceutical agenttightly bound therein may decrease the MIC (minimum inhibitoryconcentrations) against the microorganism, because the tightly-boundmolecules favorably bind to the surface of both bacteria (gram-positiveand gram-negative) and fungi. Therefore, in some embodiments the singleprotein-tightly bound pharmaceuticals can be characterized by thecomparison of their MTD or MIC to that of free form of molecules.

As described in the previous section, during the preparation process,the intended pharmaceutical molecules are entrapped in the bindingpockets of the single protein once the single protein-tightly boundpharmaceuticals are successfully prepared. Compared to the free form,the encapsulated molecules are surrounded by different environments,which could cause the changes of their UV spectra or florescenceemission spectroscopy. The carrier, binding and proximity relationshipsof the encapsulated molecules can be characterized and analyzed usingabsorption, fluorescence, FTIR, or circular dichroism.

Single Protein Encapsulation can be particularly useful withpharmaceutical agents that have limited solubility. For example, SingleProtein Encapsulation can be useful with a pharmaceutical agent thatneeds to be formulated with one or more surfactants or solubilizingcarriers to facilitate administration. In many cases, surfactants andsolubilizing carriers have undesirable properties that produce unwantedeffects upon administration. Encapsulating a pharmaceutical agent thathas limited solubility in a Single Protein can provide an administrableform of the therapeutic agent that does not include undesirablesurfactants or solubilizing carriers. Accordingly, in one embodiment,the first pharmaceutical agent and/or the second pharmaceutical agent isan agent that has poor solubility (e.g. a solubility of less than about0.1 μg/mL in water). In another embodiment, the pharmaceuticalcomposition described herein does not comprise a surfactant or asolubilizing carrier.

In one embodiment, the pharmaceutical agent is an Anthracycline, aCytoskeletal disruptor, an Inhibitor of topoisomerase I, an Inhibitor oftopoisomerase II, a Kinase inhibitor, or a Vinca alkaloid or aderivative thereof.

In one embodiment, the second pharmaceutical agent is an Anthracycline,a Cytoskeletal disruptor, an Inhibitor of topoisomerase I, an Inhibitorof topoisomerase II, a Kinase inhibitor, or a Vinca alkaloid or aderivative thereof.

In one embodiment, the pharmaceutical agent is an Anthracycline and thesecond pharmaceutical agent is a Cytoskeletal disruptor.

In one embodiment, the pharmaceutical agent is an Anthracycline and thesecond pharmaceutical agent is a Vinca alkaloid or a derivative thereof.

In one embodiment, the pharmaceutical agent is a Cytoskeletal disruptorand the second pharmaceutical agent is an Inhibitor of topoisomerase I.

In one embodiment, the pharmaceutical agent is a Cytoskeletal disruptorand the second pharmaceutical agent is an I Inhibitor of topoisomeraseII.

In one embodiment, the pharmaceutical agent is an Anthracycline and thesecond pharmaceutical agent is a kinase inhibitor.

In one embodiment, the pharmaceutical agent is an Alkylating agent, anAntimetabolite, an Anti-microtubule agent, a Topoisomerase inhibitors,or a Cytotoxic antibiotic.

In one embodiment, the second pharmaceutical agent is an Alkylatingagent, an Antimetabolite, an Anti-microtubule agent, a Topoisomeraseinhibitors, or a Cytotoxic antibiotic.

In one embodiment, the pharmaceutical agent is an Anti-microtubule agentand the second pharmaceutical agent is a Topoisomerase inhibitor.

In one embodiment, the pharmaceutical agent is an Anti-microtubule agentand the second pharmaceutical agent is a Cytotoxic antibiotic.

In one embodiment, the pharmaceutical agent is a Topoisomerase inhibitorand the second pharmaceutical agent is a Cytotoxic antibiotic.

In one embodiment, the pharmaceutical agent is an Anti-microtubule agentand the second pharmaceutical agent is an alkylating agent.

In one embodiment, a plurality of molecules of the pharmaceutical agentare tightly bound within each single protein.

In one embodiment, at least one molecule of the pharmaceutical agent istightly bound within each single protein.

In one embodiment, the composition comprises water and one or more watersoluble organic solvents.

In one embodiment, the pharmaceutical agent has poor water solubility.

In one embodiment, the maximum tolerated dose of the pharmaceuticalagent in the composition is greater than the maximum tolerated dose ofthe free form of pharmaceutical agent alone (e.g. formulated without thesingle protein).

In one embodiment, the maximum tolerated dose of the pharmaceuticalagent in the composition is at least 10% greater than the maximumtolerated dose of the free form of pharmaceutical agent alone (e.g.formulated without the single protein).

In one embodiment, the efficacy of the pharmaceutical agent in thecomposition is greater than the efficacy of the pharmaceutical agentalone.

In one embodiment, the efficacy of the pharmaceutical agent in thecomposition is at least 10% greater than the efficacy of thepharmaceutical agent alone (e.g. formulated without the single protein).

In one embodiment, the single protein, that is tightly bound to thepharmaceutical agent has a difference in absorption, fluorescence,circular dichroism spectra, or FTIR compared to a correspondingun-tightly bound single protein.

In one embodiment, one or more molecules of the second pharmaceuticalagent are tightly bound in the single protein.

In one embodiment, a plurality of molecules of the second pharmaceuticalagent are encapsulated within each single protein.

In one embodiment, at least one molecule of the second pharmaceuticalagent is encapsulated within each single protein.

In one embodiment, each molecule of the second pharmaceutical agent iscompletely encapsulated within the single protein.

In one embodiment, only part of the surface area of at least onemolecule of the second pharmaceutical agent is encapsulated by thesingle protein.

In one embodiment, the second pharmaceutical agent has poor watersolubility.

In one embodiment, the maximum tolerated dose of the secondpharmaceutical agent in the composition is greater than the maximumtolerated dose of the free form of pharmaceutical agent alone (e.g.formulated without the single protein).

In one embodiment, the maximum tolerated dose of the secondpharmaceutical agent in the composition is at least 10% greater than themaximum tolerated dose of the free form of pharmaceutical agent alone(e.g. formulated without the single protein).

In one embodiment, the efficacy of the second pharmaceutical agent inthe composition is greater than the efficacy of the pharmaceutical agentalone (e.g. formulated without the single protein).

In one embodiment, the efficacy of the second pharmaceutical agent inthe composition is at least 10% greater than the efficacy of thepharmaceutical agent alone (e.g. formulated without the single protein).

In one embodiment, the single protein that is tightly bound to thesecond pharmaceutical agent has a difference in absorption,fluorescence, circular dichroism spectra, or FTIR compared to acorresponding single protein that is not tightly bound to the secondpharmaceutical agent.

In one embodiment, the invention provides a method for treating cancerin a human or an animal comprising administering a composition of theinvention to the human or animal. In one embodiment, the therapeuticagent is doxorubicin and wherein maximum tolerated dose of thedoxorubicin in the single protein composition is at least 10% greaterthan the maximum tolerated dose of doxorubicin alone (e.g. formulatedwithout the single protein).

In one embodiment, the invention provides a method comprising: Combininga first solution that comprises: water, one or more molecules of apharmaceutical agent, and optionally one or more water soluble organicsolvents, with a second solution that comprises: water, a singleprotein, and optionally one or more water soluble organic solvents, toprovide a third solution. In one embodiment, the invention furtherprovides sterilizing the third solution via a filtration to removemicroorganisms.

The formulations of the invention may be systemically administered,e.g., orally, in combination with a pharmaceutically acceptable vehiclesuch as an inert diluent or an assimilable edible carrier. They may beenclosed in hard or soft shell gelatin capsules, may be compressed intotablets, or may be incorporated directly with the food of the patient'sdiet. For oral therapeutic administration, the formulations may becombined with one or more excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. Such compositions and preparations shouldcontain at least 0.1% of the formulation. The percentage of thecompositions and preparations may, of course, be varied and mayconveniently be between about 2 to about 60% of the weight of a givenunit dosage form.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Any material used in preparing any unit dosageform should be pharmaceutically acceptable and substantially non-toxicin the amounts employed. In addition, the formulations may beincorporated into sustained-release preparations and devices.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form should be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride.

Sterile injectable solutions are prepared by incorporating theformulations in the required amount in the appropriate solvent withvariety of the other ingredients enumerated above, as required, followedby filter sterilization. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and the freeze drying techniques, whichyield a powder of the active ingredient plus any additional desiredingredient present in the previously sterile-filtered solutions.

Useful dosages of the formulations can be determined by comparing theirin vitro activity, and in vivo activity in animal models. Methods forthe extrapolation of effective dosages in mice, and other animals, tohumans are known to the art; for example, see U.S. Pat. No. 4,938,949.

The amount of the formulation required for use in treatment will varywith the particular formulation selected, with the route ofadministration, with the nature of the condition being treated and withthe age and condition of the patient and will be ultimately at thediscretion of the attendant physician or clinician.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations.

Formulations of the invention can also be administered in combinationwith other therapeutic agents. Accordingly, in one embodiment theinvention also provides a composition comprising a formulation of theinvention, at least one other therapeutic agent, and a pharmaceuticallyacceptable diluent or carrier. The invention also provides a kitcomprising a formulation of the invention, at least one othertherapeutic agent, packaging material, and instructions foradministering the formulation and the other therapeutic agent or agentsto an animal.

The unusually long half-life of HSA or IgG is maintained mainly by FcRnor the Brambell receptor (Brambell, F. W., et el., Nature, 1964, 203,1352-1354), which is expressed in a wide variety of tissues and organs(Sockolosky, J. T., and Szoka, F. C., Adv Drug Deliv Rev, 2015, 91,109-124). This major histocompatibility complex class I-related receptorwas originally discovered to play an important role in the delivery ofIgGs from the mother to the young, regulate serum IgG concentration, andmaintain the long half-life of IgGs in the serum. It was laterdiscovered that FcRn can bind both IgG and HSA at different sites and isresponsible for the long half-lives of both IgGs and HAS (Chaudhury, C.,J Exp Med, 2003, 197, 315-322; Anderson, C. L., et el., Trends Immunol,2006, 27, 343-348; Chaudhury, C., et el., Biochemistry, 2006, 45,4983-4990; and Kim, J., Bronson, et el., Am J Physiol Gastrointest LiverPhysiol, 2006, 290, G352-360). As expected, FcRn mutation was found tocause familial hypercatabolic hypoproteinemia (Wani, M. A., et el., ProcNatl Acad Sci USA, 2006, 103, 5084-5089).

The mechanism of HSA rescue and recycling has been elucidated andinvolves:

-   -   1. FcRn binding HSA in the endosome due to high affinity at        acidic pH,    -   2. The resulting FcRn-HSA complex (1:1 ratio) is sent back to        the bloodstream,    -   3. The FcRn-HSA complex dissociates due to low affinity at pH        7.4, releasing HSA back in the circulation.        In cells that express low levels of FcRn, HSA would be        endocytosed to lysosomes, where it is degraded to amino acids.        As a result, this FcRn-mediated recycling pathway is a major        factor contributing to the long half-life of both IgGs and HSA        in human, and has significant pathophysiological and therapeutic        implications.

From publically available protein expression databases (Uhlen, M., etel., Science, 2015, 347, 1260419; Lindskog, C., Expert Rev Proteomics,2016, 13, 627-629; Tang, Z., Nucleic Acids Res, 2017, 45, W98-W102;Uhlen, M., Science, 2017, 357; and Papatheodorou, I., et el., NucleicAcids Res, 2018, 46, D246-D251), differences in FcRn expression betweennormal and cancer tissues from different human organs was investigated.A FcRn expression comparison was made in 31 different tissues. Amongthem, more than half (16/31) of cancer tissues express less FcRn thantheir normal tissue counterparts, while about more than a quarter(12/31) of cancer tissues express more FcRn, with only 3 having similarFcRn levels. The first group (16 cancers, table 1) is the specifictargets for SPE-anticancer drugs.

TABLE 1 Tumors Types Targeted for treatment (ratio of FcRn innormal/tumor tissue ≥1.19) Ratio of FcRn Tumor in Normal/ AbbreviationTumor Type Tumor tissue ACC Adrenocortical carcinoma 2.63 BLCA BladderUrothelial Carcinoma 1.81 BRCA Breast invasive carcinoma 1.66 CESCCervical squamous cell carcinoma and 3.48 endocervical adenocarcinomaCHOL Cholangiocarcinoma 1.75 DLBC Lymphoid Neoplasm Diffuse Large 1.55B-Lymphoma KICH Kidney Chromophobe 1.92 LUAD Lung adenocarcinoma 1.62LUSC Lung squamous cell carcinoma 3.14 OV Ovarian serouscystadenocarcinoma 2.30 PCPG Pheochromocytoma and Paraganglioma 1.33PRAD Prostate adenocarcinoma 1.64 SARC Sarcoma 1.19 THCA Thyroidcarcinoma 1.39 UCEC Uterine Corpus Endometrial Carcinoma 1.61 UCSUterine Carcinosarcoma 1.42

The invention will now be illustrated by the following non-limitingExamples.

EXAMPLES Example 1: Targeting Effects of SPE-Anticancer Drugs

Recognizing the problems with current cancer drug formulation/deliverysystems, biomacromolecules were investigated as highly efficient drugdelivery systems. In particular, investigations focused on: 1) usingcomponents of all natural bio-macromolecules or proteins producednaturally or by recombinant techniques and 2) avoiding chemicalmodification and conjugation.

Based on this work, an SPE (Single-Protein-Encapsulation) platform fornovel drug formulation was developed. For example, applying SPE to DOXencapsulation produces SPEDOX (single protein-encapsulated DOX), whichcontains two components, DOX and HSA. Unlike HSA-NPs that usuallycontains a number of HSA molecules through chemicalconjugation/crosslinking or aggregation, SPEDOX provides a stable anduniform molecular system comprising a single HSA molecule that enclosesa variable number of DOX molecules that can be accurately controlled.

Based on the mechanism of HSA rescue and recycling by FcRn in humans(Hoogenboezem, E. N., and Duvall, C. L., Adv Drug Deliv Rev, 2018, 130,73-89), HSA would be taken up and endocytosed to the lysosome forhydrolysis to yield peptides and amino acids by those cells that expresslow levels of FcRn. Applying this concept to the SPE drug deliveryplatform, normal cells express normal levels of FcRn that effectivelyrecycles SPE Drugs to maintain its long circulation in the body. If, onthe other hand, a type of cancer cells expresses little or no FcRn, SPEdrugs would be taken up by this type of cancer cells and endocytosed tothe lysosome, where HSA is hydrolyzed and drugs are released. Theresulting drug is then diffused out of the lysosome to the cytosol,where it is transported into the nucleus through the nuclear porecomplexes. Therefore, a cancer type that expresses little or no FcRn(table 1) presents an ideal target for treatment with SPE-drugs.

Example 2: Preparation of Composition 1 (HSA-Encapsulated Topotecan)

To 2 mL of commercial HSA solution (25 HSA) was added 2 mL of deionizedwater and 1 mL of 50% propylene glycol/water solution, in 50 mL roundbottom flask containing a magnetic stirring bar, the mixture was stirredfor 5 minutes. To a 15 mL centrifuge tube was added 25 mg of topotecanHCl salt, 1.2 mL of 50% propylene glycol/water and 3.8 mL of deionizedwater. After mixing well, the prepared yellowish solution was graduallyadded into the above HSA solution with stirring. After stirring the redmixture for 1 h at room temperature. The yellowish solution wascentrifuged at 12000 RPM for 7 min, and top solution was taken andanalyzed by UV spectrometer. The spectral changes of topotecan beforeand after encapsulation was shown FIG. 1 .

Example 3: Preparation of Composition 2 (HSA-Encapsulated Epirubicin)

To 4 mL of commercial HSA solution (25 HSA) was added 3 mL of deionizedwater and 1 mL of 56% ethanol/water solution, in 50 mL round bottomflask containing a magnetic stirring bar, the mixture was stirred for 5minutes. To a 15 mL centrifuge tube was added 72 mg of epirubicin HClsalt, 2.0 mL of 56% ethanol/water and 6 mL of deionized water. Aftermixing well, the prepared red solution was gradually added into theabove HSA solution with stirring. After stirring the red mixture for 1hours at room temperature. The red solution was centrifuged at 12000 RPMfor 10 min, and top solution was taken and analyzed by UV spectrometerand Dynamic Light Scattering instruments. The spectral changes ofepirubicin before and after encapsulation was shown FIG. 2 and particlesize distribution of composition 2 was shown in FIG. 3 .

Example 4: Preparation of Composition 3 (HSA-Encapsulated Idarubicin)

To 2 mL of commercial HSA solution (25 HSA) was added 2 mL of deionizedwater and 1 mL of 50% methanol/water solution, in 50 mL round bottomflask containing a magnetic stirring bar, the mixture was stirred for 5minutes. 1. To a 15 mL centrifuge tube was added 33 mg of idarubicin HClsalt, 3.0 mL of 50% methanol/water and 6 mL of deionized water. Aftermixing well, the prepared reddish solution was gradually added into theabove HSA solution with stirring. After stirring the reddish mixture for1 hours at room temperature, the mixture was concentrated to about 9 mLvia a high vacuum pump, the mixture was centrifuged at 4400 RPM for 10min. The composition 3 was further purified by Sephadex g 25 column. Thepurified HSA-encapsulated idarubicin was analyzed by UV spectrometer andDynamic Light Scattering instruments. The spectral changes of idarubicinbefore and after encapsulation was shown FIG. 4 and particle sizedistribution of composition 3 was shown in FIG. 5 .

Example 5: Preparation of Composition 4 (HSA-Encapsulated Vincristine)

To 2 mL of commercial HSA solution (25 HSA) was added 2 mL of deionizedwater and 1 mL of 50% methanol/water solution, in 50 mL round bottomflask containing a magnetic stirring bar, the mixture was stirred for 5minutes. To a 15 mL centrifuge tube was added 44 mg of vincristine.H₂SO₄ salt, 3.5 mL of 50% methanol/water and 6.5 mL of deionized water.After mixing well, the prepared colorless solution was gradually addedinto the above HSA solution with stirring. After stirring the mixturefor 1 h at room temperature, the mixture was concentrated to about 8 mLvia a high vacuum pump, the mixture was centrifuged at 4400 RPM for 10minutes, the resulting solution was centrifuged at 12000 RPM for 10minutes, and top solution was taken. The composition 4 was furtherpurified by Sephadex g 25 column. The purified HSA-encapsulatedvincristine was analyzed by UV spectrometer. The spectral changes ofvincristine before and after encapsulation was shown FIG. 6 .

Example 6: Preparation of Composition 5 (HSA-Encapsulated Daunorubicin)

To 2 mL of commercial HSA solution (25 HSA) was added 2 mL of deionizedwater and 1 mL of 50% methanol/water solution, in 50 mL round bottomflask containing a magnetic stirring bar, the mixture was stirred for 5minutes. To a 15 mL centrifuge tube was added 29 mg of daunorubicin HClsalt, 2.0 mL of 50% methanol/water and 6 mL of deionized water. Aftermixing well, the prepared red solution was gradually added into theabove HSA solution with stirring. After stirring the red mixture for 1hours at room temperature, the mixture was concentrated to about 7 mLvia a high vacuum pump, the resulting solution was centrifuged at 12000RPM for 10 min, and top solution was taken. The composition 5 wasfurther purified by Sephadex g 25 column. The purified HSA-encapsulateddaunorbicin was analyzed by UV spectrometer and Dynamic Light Scatteringinstruments. The spectral changes of daunorubicin before and afterencapsulation was shown FIG. 7 and particle size distribution ofcomposition was shown in FIG. 8 .

Example 7: Preparation of Composition 6 A (HSA-encapsulatedMitoxantrone)

To 3 mL of commercial HSA solution (25 HSA) was added 2 mL of deionizedwater in mL round bottom flask containing a magnetic stirring bar, themixture was stirred for 5 minutes. To a 15 mL centrifuge tube was added47 mg of mitoxantrone 2HCl salt, 3.0 mL of 50% methanol/water and 7 mLof deionized water. After mixing well, the prepared blue solution wasgradually added into the above HSA solution with stirring and. Afterstirring the blue mixture for 1 hours at room temperature, the mixturewas concentrated to about 7 mL via a high vacuum pump, the resultingsolution was centrifuged at 12000 RPM for 10 minutes, and top solutionwas taken. The composition 6 was further purified by Sephadex g 25column. The purified HSA-encapsulated mitoxantrone was analyzed by UVspectrometer and Dynamic Light Scattering instruments. The spectralchanges of mitoxantrone before and after encapsulation was shown FIG. 9and particle size distribution of composition 6 was shown in FIG. 10 .

Example 8: Preparation of Composition 7 (HSA-Encapsulated Doxorubicin)

To 2 mL of commercial HSA solution (25 HSA) was added 2 mL of deionizedwater and 1 mL of 56% ethanol/water solution, in 50 mL round bottomflask containing a magnetic stirring bar, the mixture was stirred for 5minutes. To a 15 mL centrifuge tube was added 22 mg of doxorubicin HClsalt, 2 mL of 56% ethanol/water and 8 mL of deionized water. Aftermixing well, the prepared red solution was gradually added into theabove HSA solution with stirring. After stirring the red mixture for 1hours at room temperature. The red solution was centrifuged at 12000 RPMfor 7 minutes, and top solution was taken and analyzed by UVspectrometer and Dynamic Light Scattering instruments. The spectralchanges of doxorubicin before and after encapsulation was shown FIG. 11and particle size distribution of composition 7 was shown in FIG. 12 .

Example 9: Preparation of Composition 8 (HSA-Encapsulated AmphotericinB)

To 2 mL of commercial HSA solution (25 HSA) was added 2 mL of deionizedwater and 2 mL of 50% methanol/water solution, in 50 mL round bottomflask containing a magnetic stirring bar, the mixture was stirred for 5minutes. To a 15 mL centrifuge tube was added 25 mg of amphotericin B, 2mL of methanol and 10 mL of deionized water, followed by adding 7 dropsof 1.0 M HCl solution. After mixing well, the prepared yellowishsolution was gradually added into the above HSA solution. After stirringthe red mixture for 1 hours at room temperature, the yellowish mixturewas centrifuged at 12000 RPM for 10 min, and top solution was taken. Thecomposition 8 was further purified by Sephadex g 25 column. The purifiedHSA-encapsulated amphotericin B was analyzed by UV spectrometer andDynamic Light Scattering instruments. The spectral changes ofamphotericin B before and after encapsulation was shown FIG. 13 andparticle size distribution of composition 8 was shown in FIG. 14 .

Example 10: Preparation of Composition 9 (HSA-Encapsulated Clofazimine)

To 2 mL of commercial HSA solution (25 HSA) was added 2 mL of deionizedwater and 1 mL of 56% ethanol/water solution, in 50 mL round bottomflask containing a magnetic stirring bar, the mixture was stirred for 5minutes. To a 15 mL centrifuge tube was added 27 mg of clofazimine, 2 mLof ethanol and 2 drops of acetic acid to form a dark red solution,followed by adding 6 mL of deionized water. After mixing well, theprepared dark red solution was gradually added into the above HSAsolution with stirring. After stirring the red mixture for 1 h at roomtemperature. The red mixture was centrifuged at 12000 RPM for 10minutes, and top solution was taken. The solution was furthercentrifuged at 12000 RPM for another 10 minutes. The top solution wastaken and then was analyzed by UV spectrometer and Dynamic LightScattering instruments. The spectral changes of clofazimine before andafter encapsulation was shown FIG. 15 .

Example 11: Preparation of Composition 10 (HSA-EncapsulatedMethotrexate)

To 2 mL of commercial HSA solution (25 HSA) was added 2 mL of deionizedwater and 1 mL of 50% methanol/water solution, in 50 mL round bottomflask containing a magnetic stirring bar, the mixture was stirred for 5minutes. To a 15 mL centrifuge tube was added 31 mg of methotrexate, 2.0mL of 50% methanol/water and 7 mL of deionized water. After mixing well,the prepared yellowish solution was gradually added into the above HSAsolution with stirring. After stirring the red mixture for 1 h at roomtemperature, HSA-encapsulated methotrexate was analyzed by UVspectrometer and Dynamic Light Scattering instruments. The spectralchanges of methotrexate before and after encapsulation was shown FIG. 16and particle size distribution of composition 10 was shown in FIG. 17 .

Example 12: Preparation of Composition 11 (HSA-Encapsulated Rifampicin)

To 2 mL of commercial HSA solution (25 HSA) was added 9 mL of deionizedwater and 1 mL of 50% methanol/water solution, in 50 mL round bottomflask containing a magnetic stirring bar, the mixture was stirred for 5minutes. To a 15 mL centrifuge tube was added 44 mg of rifampicin, 2.0mL of methanol and 1 mL of water. After mixing well, the prepared redsolution was gradually added into the above HSA solution with stirring.After stirring the red mixture for 2 hours at room temperature, the redmixture was centrifuged at 12000 RPM for 10 min, and top solution wastaken, which was further purified by Sephadex g 25 column. The purifiedHSA-encapsulated rifampicin was analyzed by UV spectrometer. Thespectral changes of rifampicin before and after encapsulation was shownFIG. 18 .

Example 13: Preparation of Composition 12 (HSA-Encapsulated Vinblastine)

To 2 mL of commercial HSA solution (25 HSA) was added 3 mL of deionizedwater and 6 mL of 50% methanol/water solution, in 50 mL round bottomflask containing a magnetic stirring bar, the mixture was stirred for 5minutes. To a 15 mL centrifuge tube was added 46 mg of vinblastine, 9.0mL of 50% methanol/water. After mixing well, the prepared solution wasgradually added into the above HSA solution with stirring. Afterstirring the mixture for 2 hours at room temperature, the mixture wasconcentrated to about 2 mL via a high vacuum pump. After adding another8 mL of di-water, the mixture was centrifuged at 4400 RPM for 10 min andthe top solution was taken and centrifuged at 12000 RPM for 7 min. TheHSA-encapsulated vinblastine was analyzed by UV spectrometer. Thespectral changes of vinblastine before and after encapsulation was shownFIG. 19 .

Example 14: Preparation of Composition 13 (HSA-Encapsulated Vinorelbine)

To 2 mL of commercial HSA solution (25% HSA) was added 2 mL of deionizedwater, 1 mL of methanol and 5 mL of 50% methanol/water solution, in 50mL round bottom flask containing a magnetic stirring bar, the mixturewas stirred for 5 minutes. To a 15 mL centrifuge tube was added 44 mg ofvinorelbine, 9.0 mL of 50% methanol/water. After mixing well, theprepared solution was gradually added into the above HSA solution withstirring. After stirring the mixture for 2 h at room temperature, themixture was concentrated to about 2 mL via a high vacuum pump. Afteradding another 8 mL of di-water, the mixture was centrifuged at 4400 RPMfor 10 min and the top solution was taken and centrifuged at 12000 RPMfor 8 min. The HSA-encapsulated vinorelbine was analyzed by UVspectrometer. The spectral changes of vinblastine before and afterencapsulation was shown FIG. 20 .

Example 15: Preparation of Composition 14 (HSA-Encapsulated Paclitaxel)

To 2 mL of commercial HSA solution (25 HSA) was added 6 mL of deionizedwater in 50 mL round bottom flask containing a magnetic stirring bar,followed by 3 mL of methanol and 5 mL of 50% methanol/water solution,the mixture was stirred for 5 min. To a 15 mL centrifuge tube was added40 mg of paclitaxel, 3.0 mL of methanol and 7 mL of 50% methanol/water.After mixing well, the prepared solution was gradually added into theabove HSA solution with stirring. After stirring the mixture for 1 h atroom temperature, the mixture was concentrated to about 2 mL via a highvacuum pump. After adding another 8 mL of di-water, the mixture wascentrifuged at 12000 RPM for 10 min, and top solution was taken, whichwas further purified by Sephadex g 25 column.

Example 16: Dialysis Study on the Free Form of Doxorubicin, HSA/DOXMixture and Composition 7

To a 3×3 cm flat dialysis tube (molecular weight cut: 25 kD) was added ared solution of 3 mg of free form of doxorubicin, 3 mg of doxorubicinequivalent of HSA/DOX mixture or 3 mg of doxorubicin equivalent ofcomposition 7 in 2 mL of di-water. The dialysis tube was sealed and putinto a glass beaker containing 100 mL of either PBS buffer (pH=7.4) oracetate buffer (pH=5.5) with a magnetic stirring bar. With constantstirring, aliquots (500 uL) at different time points were taken from theglass beaker and analyzed by UV spectrometer. The percent of thedialyzed out from the dialysis tubes at different pH values wascalculated and shown in FIG. 22 . It is very clear that HSA/DOX mixturewas dialyzed out almost 100% at pH=7.4 within 24 h; for free form ofdoxorubicin at pH=7.4, it was dialyzed out very fast in the earlier timeand but it precipitated in the dialysis bag due to the poor solubilityat pH=7.4. It is most interesting to notice that HSA-encapsulated DOX isalmost 3.5 times faster at pH=5.5 than at pH=7.4 for being dialyzed outfrom the dialysis tube.

Example 17: Efficacy Study

An efficacy study against breast cancer tumor was carried out on thematerials from Example 8 (Composition 7) and Example 7 (Composition 6).It is documented that the maximum tolerated dose (MTD) of free DOX in amouse model is 5 mg/kg and the MTD of free mitroxantrone is 3 mg/kg. Theresults of a xenograft model efficacy study using breast cancer cellline, MDA-MB231, in female athymic nude mice, free DOX as 5 kg/mL, andmaterial from Example 7 as 10 mg/kg (DOX equivalent, 2× of MTD of thefree DOX), material from Example 6, as 6 mg/kg (mitoxantrone equivalent,2× of MTD of the free mitoxantrone) are shown in 1, FIG. 23 and FIG. 24. For doxorubicin, HSA-tightly bound Dox, is significantly better thanfree DOX in inhibiting tumor growth (p<0.05); with % TGI at 54% for thematerial from sample 7 vs 34 for free DOX. For mitoxantrone, HSA-tightlybound mitoxantrone, is significantly much better than free mitoxantronein inhibiting tumor growth (p<0.01); with % TGI at 58% for the materialfrom sample 6 vs 27 for free mitoxantrone. Furthermore, by comparing thebody weight changes (normalized), shown in FIG. 25 and FIG. 26 ,Composition 7, though 2× of DOX equivalents, did not show any toxicity(FIG. 25 ), however; Composition 6 from Example 7, though 2× ofmitoxantrone equivalents, show some acceptable toxicity (only average10% body weight loss, see FIG. 26 ). Results are shown in Table 2 forrepresentative compositions.

TABLE 2 Tumor Growth Inhibition in MDA-MB-231 Study using Composition 6and Composition 7 Treatment Regimen MTV (n) Statistical SignificanceRegressions Mean BW Deaths Group n Agent mg/kg Route Schedule Day 20 %TGI vs G1 vs G3 vs G5 PR CR Nadir TR NTR 1 8 vehicle — iv qwk × 3 683(8) — — — — 0 0 — 0 0 2 8 Doxorubicin 5 iv qwk × 3 453 (8) 34 ns ns — 00 — 0 0 3 8 Composition 7 10 iv qwk × 3 311 (8) 54 * — — 0 0 — 0 0 4 8Mitoxantrone 3 iv qwk × 3 498 (8) 27 ns — * 0 0 −0.2% Day 8 0 0 5 7Composition 6 6 iv qwk × 3 288 (7) 58 ** — — 0 0 −8.7% Day 8 0 1 TheabovetTable displays the final treatment regimen at completion of thestudy, vehicle = Sterile Water Study Endpoint = 2000 mm³; Study Duration= 20 Days n = number of animals in a group not dead from accidental orunknown causes, or euthanized for sampling % TGI =[1-(MTV_(drug treated)/MTV_(control)] × 100 = percent tumor growthinhibition, compared to Group 1 Statistical Significance (Mann-Whitney Utest): ne = not evaluable, ns = not significant, * = P < 0.05, ** = P <0.01, *** = P < 0.001, compared to Group 1 MTV (n) = median tumor volume(mm²) for the number of animals on the Day of TGI analysis (includesanimals with tumor volume at endpoint) PR = partial regression; CR =complete regression Mean BW Nadir = lowest group mean body weight, as %change from Day 1; — indicates no decrease in mean body weight wasobserved TR = treatment-related death; NTR = non-treatment-related death

Example 18: Toxicity Study

In order to investigate toxicity of Composition 7, a MTD study usinghealth female athymic nude mice (4 mice per group) with the same routeand schedule like the efficacy study (Example 13), was conducted andcompleted. The resulting data is shown in FIG. 27 . These datademonstrate that 3 times and 4 times of DOX equivalents in the materialfrom Composition 7 did not show toxicity.

Example 19: Second MTD Study

A second MTD study using the same protocols, with varied doses was alsocompleted. The resulting data is presented in FIG. 28 . These datademonstrate that 5 times and 6 times of DOX equivalents in Composition 7are still very safe.

Example 20: Preparation of Composition 15 (IGG-Encapsulated Doxorubicin)

To 35 mg of doxorubicin hydrochloride was added 10 mL of Deionizedwater, after shaking well, the red solution was formed. Into thissolution was added 500 mg of IgG antibody powder, the mixture wasstirred for 1 hour and the red solution was prepared. And then 4.0 mL of50% ethanol/H₂O was added. The mixture was stirred for 1 h and mixturewas centrifuged at 4400 RPM for 12 minutes, the top solution was takenand the pH was adjusted 7.3 by adding 0.5 M HCl solution. TheIgG-encapsulated doxorubicin (composition 15) was analyzed by UVspectrometer. The spectral changes of doxorubicin before and afterencapsulation was shown in FIG. 21 .

Example 21: Preparation of Composition 16 (IGG-Encapsulated Epirubicin)

To 28 mg of epirubicin hydrochloride was added 12 mL of Deionized water,after shaking well, the red solution was formed. Into this solution wasadded 500 mg of IgG antibody powder, the mixture was stirred for 1 hourand the red solution was prepared. And then 5.0 mL of 50% ethanol/H₂Owas added. The mixture was stirred for 1 hour and mixture wascentrifuged at 4400 RPM for 12 minutes, the top solution was taken. TheIgG-encapsulated epirubicin (composition 16) was analyzed by UVspectrometer. The spectral changes of doxorubicin before and afterencapsulation was shown FIG. 33 .

Example 22: Preparation of Composition 17 (HSA-Encapsulated Docetaxel)

To 6.5 mL of commercial HSA solution (25 HSA) was added 12.5 mL ofdeionized water and 8.5 mL of 50% tert-butanol/water solution, in 50 mLround bottom flask containing a magnetic stirring bar, the mixture wasstirred for 5 minutes. To a 15 mL centrifuge tube was added 71 mg ofdocetaxel, 3.0 mL of ethanol. The resulting docetaxel solution was addedinto the above HSA. After stirring the mixture for 2 hours at roomtemperature, the mixture was centrifuged at 4400 RPM for 10 minutes andthe top solution was taken. After lyophilizing, HSA-encapsulateddocetaxel (composition 17) powder was obtained.

Example 23: Preparation of Composition 18 (HSA-Encapsulated Doxorubicinand Docetaxel)

To 66 mg of doxorubicin hydrochloride was added 14 mL of Deionizedwater, after shaking well, the red solution was formed. Into thissolution was added 1.6 g of HSA powder, the mixture was stirred for 20minutes and then 6.0 mL of 50% ethanol/H₂O was added. The mixtures wasstirred for 30 minutes. In another 15 mL of centrifuge tube was added 46mg of docetaxel and 3 ml of ethanol. The resulting docetaxel solutionwas added into the above red solution. The mixture was stirred for 2hours and mixture was centrifuged at 4400 RPM for 10 minutes, the topsolution was taken The HSA-encapsulated doxorubicin and docetaxel(composition 18) was analyzed by UV spectrometer. The spectral changesof doxorubicin before and after encapsulation was shown FIG. 34 . Afterlyophilizing, the composition 18 powder form was obtained.

All publications, patents, and patent documents are incorporated byreference herein, as though individually incorporated by reference. Theinvention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

1. A composition comprising, a single protein selected from the groupconsisting of an albumin, a globulin, and a fibrinogen having one ormore molecules of an anticancer agent other than doxorubicin tightlybound therein. 2-21. (canceled)
 22. The composition of claim 1, whereinthe single protein is an albumin.
 23. The composition of claim 1,wherein the single protein is a globulin.
 24. The composition of claim1, wherein the single protein is a fibrinogen.
 25. The composition ofclaim 1, wherein the single protein is IgA, IgM, or IgG.
 26. Thecomposition of claim 1, wherein a plurality of molecules of theanticancer agent are tightly bound within each single protein.
 27. Thecomposition of claim 1, wherein each molecule of the anticancer agent iscompletely encapsulated within the single protein.
 28. The compositionof claim 1, wherein only part of the surface area of at least onemolecule of the anticancer agent is encapsulated by the single protein.29. The composition of claim 1, which comprises water and one or morewater soluble organic solvents.
 30. The composition of claim 1, whereinthe anticancer agent is an anthracycline, a cytoskeletal disruptor, anInhibitor of topoisomerase I, an Inhibitor of topoisomerase II, a kinaseinhibitor, a vinca alkaloid, an alkylating agent, an antimetabolite, ananti-microtubule agent, a topoisomerase inhibitors, or a cytotoxicantibiotic.
 31. The composition of claim 1, wherein the anticancer agentis mitoxantrone, daunorubicin, epirubicin, idarubicin, vincristine,vinorelbine, vinblastine, topotecan, irinotecan, actinomycin D,idarubicin, methotrexate, pemetrexed, raltitrexed, SN-38, ixabepilone,eribulin, vindesine, camptothecin, taxol, docetaxel, bendamustine,nelarabine, pirarubicin, clofarabine, valrubicin, chlorambucil, ormelphalan.
 32. The composition of claim 1, wherein the anticancer agentis actinomycin D, SN-38, or docetaxel.
 33. The composition of claim 1,wherein the maximum tolerated dose of the anticancer agent in thecomposition is at least 10% greater than the maximum tolerated dose ofthe free form of anticancer agent alone.
 34. The composition of claim 1,wherein the efficacy of the anticancer agent in the composition is atleast 10% greater than the efficacy of the anticancer agent alone. 35.The composition of claim 1, wherein the single protein, that is tightlybound to the one or more molecules of an anticancer agent has adifference in absorption, fluorescence, circular dichroism spectra, orFTIR compared to a corresponding un-tightly bound single protein.
 36. Acomposition, comprising a single protein selected from the groupconsisting of an albumin, a globulin, and a fibrinogen having tightlybound therein, a plurality of molecules of an anticancer agent otherthan doxorubicin.
 37. The composition of claim 36, wherein theanticancer agent is actinomycin D, SN-38, or docetaxel.
 38. Thecomposition of claim 36, wherein the maximum tolerated dose of theanticancer agent in the composition is at least 10% greater than themaximum tolerated dose of the free form of anticancer agent alone.
 39. Acomposition, comprising a single protein selected from the groupconsisting of an albumin, a globulin, and a fibrinogen having one ormore molecules of an anticancer agent other than doxorubicin completelyencapsulated therein.
 40. The composition of claim 39, wherein theanticancer agent is actinomycin D, SN-38, or docetaxel.